INFECTION AND IMMUNITY, Jan. 1990, p. 80-87

Vol. 58, No. 1

0019-9567/90/010080-08$02.00/0 Copyright © 1990, American Society for Microbiology

A Major Immunogenic 36,000-Molecular-Weight Antigen from Mycobacterium leprae Contains an Immunoreactive Region of Proline-Rich Repeats JELLE E. R. THOLE, LINDA F. E. M. STABEL, MARJON E. G. SUYKERBUYK, MADELEINE Y. L. DE WIT, PAUL R. KLATSER, AREND H. J. KOLK, AND RUDY A. HARTSKEERL* N. H. Swellengrebel Laboratory of Tropical Hygiene, Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands Received 10 July 1989/Accepted 29 September 1989

The 36,000-molecular-weight antigen (36K antigen) of Mycobacterium leprae is a major immunogenic protein carrying common and specific antigenic determinants recognized by antibodies and T cells in leprosy patients. Recombinant DNA clones containing the complete gene coding for the 36 K antigen, designated in this paper as PRA, were isolated from both lambda gtll and cosmid libraries of the M. kprae genome. The DNA sequence of the pra gene coded for a polypeptide of 249 amino acids with a predicted molecular mass of 26,299 daltons. The deduced amino acid sequence revealed a proline-rich (42%) amino-terminal region containing a number of repeated sequences similar or identical to the sequence PGGSYPPPPP. The reactivity of four monoclonal antibodies (F47-9, F67-1, F67-5, and F126-5) was directed to this proline-rich region of the PRA protein. DNA sequence and immunological data indicated that the lambda gtll recombinant Y3180, which was previously isolated by using antibody F47-9 (R. A. Young, V. Mehra, D. Sweetser, T. Buchanan, J. Clark-Curtiss, R. W. Davis, and B. R. Bloom, Nature (London) 316:450-452, 1985), specffies a fusion protein unrelated to PRA but containing a similar epitope recognized by F47-9.

Although Mycobacterium leprae was one of the first human pathogens to be described, relatively little is known about the components that play a role in the immunopathology of leprosy. The availability of purified and well-characterized antigens is a prerequisite for the determination of the role of individual molecules in the pathogenesis of leprosy and in the humoral and cellular immune responses to M. leprae and for establishing their potential use as diagnostic reagents or vaccine components. However, until recently, the inability to grow M. leprae in vitro and the relatively complex biochemical structure of the bacterium have severely hampered the isolation and characterization of individual antigenic components. Despite these problems a number of protein, carbohydrate, and lipid antigens of M. leprae have been isolated and their roles in immune reactions to M. leprae have been studied (13, 38). A well-known example is phenolic glycolipid I, which was shown to be useful for serodiagnostic purposes and which seems to play a role in antigen-specific suppression of cell-mediated immunity in lepromatous leprosy patients (6, 21). The recent availability of monoclonal antibodies and the construction of genomic libraries of M. leprae have facilitated the characterization and expression of genes specifying M. leprae protein antigens (8, 11, 40). Recombinant DNA clones expressing 12,000-molecularweight (12K), 18K, 28K, 36K, 65K, and 70K antigens in Escherichia coli K-12 have been selected from a lambda gtll library, and the genes for the 18K, 28K, 65K, and 70K proteins have been characterized (2, 5, 12, 22, 40). We have previously reported the isolation and characterization of a protein antigen with an apparent molecular weight of 36,000 which carries an M. leprae-specific antigenic determinant recognized by the monoclonal antibody F47-9 (10, 17). This antibody has been used to develop a competition enzyme*

linked immunosorbent assay (ELISA) for serological investigation of leprosy patients (15, 16). T cells from tuberculoid leprosy patients recognized both common and specific epitopes on this antigen (25, 36). Moreover, the 36K antigen was shown to play a regulatory role in the cellular immune responses of leprosy patients, since M. leprae-specific suppressor T cells isolated from a borderline lepromatous leprosy patient were found to suppress the response of helper T cells to the 36K antigen (24). In this paper, we describe the molecular cloning and characterization of the 36K antigen of M. leprae, as well as the characterization of recombinant phage Y3180 (40), expressing an antigenic determinant recognized by monoclonal antibody F47-9. As the DNA sequence revealed a high content of prolines, the 36K protein is referred to in this paper as proline-rich antigen (PRA). MATERIALS AND METHODS

Bacterial strains, bacteriophages, and plasmids. The bacterial strains, phages, and plasmids used in this study are listed in Table 1. Strain PC2495 was used as a host for plasmids pEMBL8 and pEMBL9 and derivatives, strain POP2136 was used as a host for plasmid pEX2 and derivatives, and strain M1070 was used for pPLc236 and derivatives. Bacteriophage lambda gtll recombinants were propagated on strain Y1090, and lysogens were constructed by using strain Y1089 as a host. Media and reagents. LB medium and LB agar were used for growing E. coli K-12 strains (20). Strain PC2495 was grown on minimal medium (9). Ampicillin and kanamycin were added as previously described (14). Restriction endonucleases and T4 DNA ligase (Boehringer GmbH, Mannheim, Federal Republic of Germany, and New England BioLabs, Inc., Beverly, Mass.) were used as specified by the manufacturers. Anti-p-galactosidase was from Promega Biotec, Madison, Wis., and horseradish peroxidase-labeled anti-

Corresponding author. 80

VOL. 58, 1990

M. LEPRAE PROLINE-RICH ANTIGEN

81

TABLE 1. Bacterial strains, phages, and plasmids Bacterial strains,

R.

Strains M. leprae 5-3 E. coli K-12 Y1089 Y1090 PC2495 POP2136 M1070

Reference or

oelevant ongin property(es

phages, and plasmids

Isolated from armadillo liver tissue

This study

AlacU169 Alon HfAISO(pMC9) AlacU169 Alon supF(pMC9) JM101 hsdR recA cI857 recA hsrK hsmK (pCI857)

R. Young (39) R. Young (39) PC,' P. Weisbeek A. Raibaud J. Thole (33)

Phages Lambda gtll THL2004 Y3180

lambda gtll recombinant containing pra lambda gtll recombinant encoding an M. leprae 36K antigenic determinant

R. Young (40) This study R. Young (40)

Plasmids pCI857 pMC9 pEMBL8 and pEMBL9 pEX1, pEX2, and pEX3 pTHL1007 pPLc236 pTHL1019 pTHL1020 pTHL1018

Apr, pBR322-lacI Apr Apr, contains PR promoter of bacteriophage lambda and cro-lacZ gene fusion pEX2 recombinant containing pra Apr, contains PL promoter of bacteriophage lambda pPLc236 recombinant containing a 3.0-kb M. leprae DNA fragment comprising pra pTHL1019 carrying the 3.0-kb DNA fragment in the opposite orientation Tcr, pHC79 recombinant containing pra

Kmr

28 4 P. Weisbeek (9) K. Stanley (32) This study E. Remaut (27) This study This study This study

a Kmr, Kanamycin resistance; Apr, ampicillin resistance; Tcr, tetracycline resistance. bPC, Phabagen Collection, Department of Molecular Cell Biology, Section Microbiology, State University of Utrecht, The Netherlands.

mouse immunoglobulin G was from DAKOPATTS Laboratories, Copenhagen, Denmark. Murine monoclonal antibodies. Monoclonal antibody F47-9 was produced as previously described (16, 18). Hybridomas producing monoclonal antibodies F67-1 and F67-5 were obtained after immunization of BALB/c mice with M. tuberculosis, and a hybridoma producing antibody F126-5 was obtained after immunization of BALB/c mice with Mycobacterium kansasii (A. Kolk et al., manuscript in preparation). F47-9 recognizes an M. leprae-specific determinant, whereas F67-1, F67-5, and F126-5 recognize broadly cross-reactive determinants on PRA. Cross-competition of monoclonal antibodies was determined by ELISA, using peroxidaselabeled antibodies as described previously (10). The conjugation of monoclonal antibodies with horseradish peroxidase was done by the procedure of Wilson and Nakane (37). DNA technology. Standard procedures were used for the preparation of phage and plasmid DNA, restriction enzyme digestion, ligation, transformation, and transduction (20). DNA sequencing of the pEX2 and pPLc236 derivatives was performed on CsCl-purified plasmid DNAs by the dideoxy chain termination method (29) using the pUC sequencing kit (Boehringer), the Sequenase sequencing kit (United States Biochemical Corp., Cleveland, Ohio), and the T7 sequencing kit (Pharmacia LKB, Uppsala, Sweden) according to the instructions of the manufacturers. Synthetic oligonucleotide primers used for DNA sequencing are indicated in Fig. 1. In addition, primers 8625 and 17588 were used. These primers are complementary to the DNA sequence 12 to 26 base pairs (bp) upstream (primer 8625) and 14 to 30 bp downstream (primer 17588) of the EcoRI site of pEX2. All primers were synthesized by H. M. Hodemaekers, National Institute of Public Health and Environmental Hygiene, Bilthoven, The Netherlands. Sequencing of the 177-bp EcoRI-BglII M. leprae DNA fragment adjacent to the lacZ gene of lambda gtll recombinant Y3180 was performed by the dideoxy chain termination method, using the vectors pEMBL8 and

pEMBL9 (9). To sequence both strands, the 177-bp EcoRIBgIII fragment was subcloned into EcoRI-BamHI digested plasmids pEMBL8 and pEMBL9, resulting in plasmids pTHL1002 and pTHL1003, respectively. Single-stranded DNA from the latter two plasmids was isolated by the method of Dente et al. (9). Southern blotting (31) and hybridization were basically done as described by Van Eys et al. (35). Preparation of 32P-labeled DNA probes was done using a random priming DNA labeling kit (Boehringer) according to the instructions of the manufacturer. Screening of lambda gtll library. A pool of the monoclonal antibodies (dilution of ascitic fluids) F47-9 (1:3,000), F67-1 (1:2,000), F67-5 (1:2,000), and F126-5 (1:500) was used to screen a lambda gtll genomic library of M. leprae constructed by Young et al. (40), for the selection of recombinants expressing antigenic determinants of PRA. Approximately 5,000 PFU per 90-mm plate of the lambda gtll library, propagated on strain Y1090, were screened for expression of recombinant PRA. Induction of expression and subsequent plaque ELISA were done as described by Young et al. (40) and Van Embden et al. (34), respectively. Screening of cosmid library. Colony hybridization (20) was used to select recombinant DNA clones containing the pra gene from the pHC79 library of the M. leprae genome constructed by Clark-Curtiss et al. (8). Approximately 500 colonies per 90-mm plate were screened, using the 32Plabeled 1.0-kilobase-pair (kbp) EcoRI M. leprae DNA fragment from lambda gtll recombinant clone THL2004 as a probe. Subcloning of pra. The hybrid plasmid pTHL1007 was constructed by subcloning the 1.0-kbp EcoRI M. leprae DNA fragment from lambda gtll recombinant clone THL2004 into the EcoRI site of the expression vector pEX2. Four deletion mutations were generated as follows (Fig. 2): digestion of pTHL1007 with the restriction endonuclease SmaI, followed by ligation at a low DNA concentration (5 ,ug/ml), resulted in plasmid pTHL1022; the 0.55-kbp HaeIII

82

THOLE ET AL. A

lty-

INFECT. IMMUN. Detail

Ecoal 3stKUl

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FIG. 1. Physical map of the 3.0-kb M. Ieprae EcoRI DNA fragment of plasmid pTHL1019 (A) and strategy of sequencing the pra gene with oligonucleotide primers (B). The regions constituting pra in recombinants pTHL1019 and pTHL1007 were sequenced. The broken line of 17588 indicates that this part of the strand was sequenced only from pTHL1007.

DNA fragment of pTHL1007 containing the 3' part of cro-lacZ fused to the 5' part of the 1.0-kbp EcoRI M. leprae DNA fragment was subcloned into the SmaI site of pEX2, resulting in plasmid pTHL1023; subcloning of the 0.25-kbp SmaI DNA fragment of pTHL1007 into the SmaI site of pEX3 resulted in plasmid pTHL1035; finally, the 0.6-kbp SmaI-BamHI DNA fragment of pTHL1007 was subcloned into the SmaI-BamHI sites of pEX1, resulting in plasmid pTHL1036. Production of recombinant PRA proteins. Expression of cro-lacZ-pra hybrid genes and the pra gene from plasmids pTHL1007, pTHL1019, pTHL1020, pTHL1022, pTHL1023, pTHL1035, and pTHL1036 was induced as described previously (41). Production of recombinant PRA protein was

analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western (immuno-) blotting. SDS-PAGE was done as described by Laemmli (19). Western blotting was done by the method of Burnette (3), as modified by Van Embden et al. (34). Mapping of B-cell epitopes. Reactivity of the monoclonal antibodies F47-9, F67-1, F67-5, and F126-5 with recombinant fusion proteins was determined by ELISA, using lysates of POP2136, harboring various plasmids, blotted onto nitrocellulose (33) and by Western blot analysis. Skin tests. Female outbred guinea pigs (Hartley-Duncan), each weighing approximately 400 g, were sensitized 28 days prior to challenge by injecting 2 x 109 whole M. leprae cells intraperitoneally. The flanks of the guinea pigs were shaved,

ostXil

pTHL1007 crolacZ

pTHL1022 pTHL1035 I

pTH1023 19

pTIL1036 pEX1,2,3

REACTIVITY VITH MONOCLONAL ANTIBODIES F47-9

F67-1

F67-5

+

+

+

F126-5 +

++

951

13

1

103

249

Epi tope

19-95

+

4

19-95

19-95

19-95

FIG. 2. Physical map of recombinant plasmids expressing various regions of the M. leprae pra gene and reactivity of monoclonal antibodies to proteins expressed by these plasmids. (Left) Fli, Vector part of the recombinant DNA; M, pra gene; PR, right lambda promoter present in the vectors pEX1, pEX2, and pEX3; -*, direction of transcription. The numbers in the pra gene indicate the amino acid residues in the PRA protein sequence which delimit the deletions. The amino-terminal methionine was taken as residue no. 1 in the PRA sequence, which has a length of 249 amino acid residues. (Right) Reactivity of monoclonal antibodies to total protein from induced POP2136 cells harboring various plasmids, established by dot blot and Western blot analysis.

VOL. 58, 1990

):, ,,%

FIG. 3. Western blot of M. leprae sonic extract and recombinant E. coli lysates, using monoclonal antibody F47-9. Proteins were separated by electrophoresis on 13% (A) and 8% (B and C) SDSpolyacrylamide gels. Antibodies F67-1, F67-5, and F126-5 gave the same reactivity patterns. (A) M1070 cells carrying pTHL1019, grown at 42°C (lane 1) and 30°C (lane 2) and M. leprae sonic extract (lane 3); (B) induced POP2136 cells carrying pTHL1007 (lane 4), pTHL1022 (lane 5), pTHL1036 (lane 6), pTHL1023 (lane 7), pTHL1035 (lane 8), and pEX2 (lane 9); (C) Y3180 lysogenized in Y1089 (lane 10) and lambda gtll lysogenized in Y1089 (lane 11). No differences were detected between the reactivities of the four monoclonal antibodies to the fusion proteins expressed by THL2004 and pTHL1007. The numbers at the left of the panels indicate molecular mass standards in kilodaltons.

and as skin test antigens, 10 ,ug of M. leprae sonic extract, recombinant PRA fusion protein, and Cro-3-galactosidase were injected intradermally. After 24 h, the diameters of the skin test reactions were determined in millimeters. The recombinant PRA fusion protein and Cro-,3-galactosidase were obtained from induced cultures of strain POP2136 carrying plasmids pTHL1007 and pEX2, respectively. The proteins were purified by preparative SDS-PAGE as described previously (10).

RESULTS Selection and characterization of recombinant phages expressing PRA antigenic determinants. Approximately 106 plaques from the lambda gtll library of M. leprae were screened for expression of antigenic determinants of PRA, using a pool of the anti-PRA monoclonal antibodies F47-9, F67-1, F67-5, and F126-5. Four positive recombinant clones were isolated and characterized with respect to DNA inserts and expression products. Recombinant THL2004 contained an insert of 1.0 kbp and expressed a fusion protein with an apparent- molecular weight of 143,000. The fusion protein reacted with all four monoclonal antibodies used in the screening (Fig. 3). The three other positive phage clones were identical to THL2004 with respect to the size of the DNA insert, the apparent molecular weight of the expressed fusion protein, and the reactivity of the four monoclonal antibodies to the expressed fusion protein. Selection of recombinants containing the complete pra gene. The size of the fusion protein expressed by the lambda gtll recombinant THL2004 suggests that this recombinant encodes about 85% of PRA. Therefore, we screened a cosmid library of the M. leprae genome to select recombinants containing the complete pra gene. Using the 1.0-kbp EcoRI

M. LEPRAE PROLINE-RICH ANTIGEN

83

insert of THL2004 as a probe, we screened approximately 1,500 colonies from the cosmid library. Of 15 positive cosmid recombinants, 9 were characterized by restriction enzyme analysis and were found to contain DNA inserts varying in size from 41 to 47 kbp. Analysis by Southern blotting revealed that all nine cosmid clones contained a 3.0-kbp EcoRI fragment that hybridized with the 1.0-kbp EcoRI fragment from lambda gtll recobinant THL2004 (results not shown). The 3.0-kbp EcoRI fragment of cosmid recombinant pTHL1018 was subcloned into the EcoRI site of the expression vector pPLc236 in both orientations, resulting in the recombinants pTHL1019 and pTHL1020. A physical map of plasmid pTHL1019 is shown in Fig. 1. Comparison with the restriction map of the 1.0-kbp EcoRI fragment from THL2004 (Fig. 2) indicated that the PRAencoding DNA sequence was localized approximately between the XmaIII and BamHI sites on the map of pTHL1019 and that the pra gene was positioned in the correct orientation with respect to the PL promoter. Expression of the pra gene in M1070 carrying plasmids pTHL1019 and pTHL1020 was analyzed by Western blotting. Induction of the thermoinducible PL promoter by incubating E. coli carrying plasmid pTHL1019 at 420C resulted in the production of a low but clearly detectable amount of PRA protein that migrated at the same position as the corresponding M. leprae antigen (Fig. 3, lane 1). No detectable amount of antigen was produced at 30°C, at which temperature the PL promoter is repressed (Fig. 3, lane 2). Cells carrying plasmid pTHL1020 did not produce antigen either at 30 or 420C (results not shown). These results indicate that the expression of the pra gene on pTHL1019 occurred from the lambda promoter and that this plasmid contains the complete pra gene. DNA sequence of the pra gene. A set of synthetic oligonucleotides was used to sequence the pra regions of recombinants pTHL1019 and pTHL1007 (Fig. 1). Plasmid pTHL1007 was constructed by subcloning the 1.0-kbp EcoRI M. leprae DNA fragment of recombinant phage THL2004 into the EcoRI site of expression vector pEX2 (Fig. 2). The DNA sequence of 1,275 bp of the region located between the XmaIII and BamHI sites of pTHL1019 is depicted in Fig. 4. The sequence of the 1.0-kbp EcoRI fragment from pTHL1007 was completely homologous to part of the sequence of pTHL1019 starting at position 359 and ending at position 1275. It was concluded that a homotogous M. leprae chromosomal sequence is present in recombinants pTHL1007 and pTHL1019, derived from two different gene libraries of M. leprae. An open reading frame corresponding to the reading frame and to the' direction of transcription of the pra gene on recombinant plasmid pTHL1007 was found beginning at position 328 and ending at position 1114. Putative translation initiation codons at the 5' end were found at position 367 and next at position 730. As the translated sequence from the latter putative start codon is only 128 amino acids long, we concluded that the translation of the pra gene starts at the ATG initiation codon at position 367. A potential Shine-Dalgarno sequence, GGAAGG, is present 12 bp upstream of this ATG start codon. Thus, the PRA protein is composed of a polypeptide of 249 amino acids with a calculated molecular mass of 26,299 daltons, which is 'considerably lower than the apparent molecular weight of 36,000 estimated from SDS-PAGE analysis. The sequence data indicate that both pTHL1019 and pTHIL1007 contain the complete coding region for PRA.

Mapping of B-cell epitopes on PRA. The binding of F67-5 to the Cro-p-galactosidase-PRA fusion protein could be inhib-

84

INFECT. IMMUN.

THOLE ET AL.

Xma III

CGGCCGTGTTTAAGGTCGTGCCAGCTTGGCCGACGCCGTCTCGGAGCACATGGGCCCGCC 60 120

CGATGCGCTGATGGTGGTGGAGAAAGGCAAACTGGTCGGGGTCATAACGCGATACGACTT

180

GTTGGGTTTCCTCTCGGAGGGCGCACCGCTACGTTAGCGGGATGATCGAGCCGCAAGTTA 240 GTTCGGCTACAAACCAAACACGGCGACGACTCGATACGCCGAAACCCGTGCCCGTTCGCT 300 SD ACGCGAATTTGTTCTCAGGTAGGGTAATGCGGCCTAGTTCAGCTCCTCCGCAGT5GAAG L 360 TTACCCATGACOGATCAACCGCCGCCGAGCGGGTCTAACCCGACACCTGCTCCGCCCCCG , 420

240

Sma

Y T D Q

P

P

P

8 G

8 N P T

P

A

P

P

CCGCCGCCTACTGCGCCACCCGTCGGTGGTTCTTACCCGCCACCTCCGCCGCCCGGCGGT 540 P P P T A P P V G G S Y P P P P P P G G

TCTTACCCGCCACCTCCGCCGCCCGGCGGTTCTTACCCGCCACCTCCGCCTTCCACCGGC Goo S Y P P P P P P G G 8 Y P P P P P S T G Hae III GCGTA0CG0GCGCCTCCACCTGGACCGGCGATCCGCTCACTGCCTAAGGAGG QTACACA A Y A P P P P G P A I R S L P K F A Y T Saa I TTCTGGGTCACCGGGCTGGCTTATGTCATCGACAACATCCCAGCCACGGTCCTGCTC PF V T R V L A Y V I D N I P A T V L L

GGCATTGGCATGTTGATTCAGACGCTCACGMGCAAGAGGCGTGCGTCACTGATATCACG I

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CAGTACAATGTTMTCAGTACTGTGCTACTCAGCCTACCGGCATOGGCATGTTGGOGTTC I---

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Y3180 fusion protein

S

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FIG. 5. Nucleotide sequence and deduced amino acid sequence of part of the M. Ieprae DNA insert of recombinant phage Y3180 (A) and comparison of the homologous regions of the PRA protein and the fusion protein encoded by Y3180 (B). Identical amino acids are indicated by a vertical line, and equivalent amino acids are indicated by a colon. The numbers at the left and right indicate the positions of the amino acids in the corresponding proteins. The amino acids of the ,B-galactosidase part of the fusion protein encoded by Y3180 are numbered -4 to -1, and the amino acids of the adjacent 36K protein part are numbered 1 to 10.

GCCACGCGCTCCGCATTOGGCATGTGTTGAGTGGTTCAGGGTGATCAGCGAGGCTACT 960 A

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1200

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1260

Bam HI

CTATCGTCCGGATCC

FIG. 4. Nucleotide and deduced amino acid sequences of the coding region of the pra gene. The putative Shine-Dalgamo (SD) sequence is underlined. Only relevant restriction enzyme sites are indicated.

ited by monoclonal antibodies F47-9 and F126-5 (Table 2). Binding of antibody F47-9 was inhibited only by F126-5, whereas binding of F126-5 was not inhibited by the other two antibodies. These results indicate that these three antibodies recognize overlapping or closely linked antigenic determinants. To localize the B-cell epitopes of PRA, four deletion mutations were generated from pTHL1007 (Fig. 2). DNA sequence analysis revealed that in all four deletion mutations the pra part was fused to cro-lacZ in the proper reading frame. After induction of the PR promoter preceding the gene fusions of pTHL1007 and the four deletion derivatives, TABLE 2. Cross-inhibition of reactivity of monoclonal antibodies to PRA fusion protein Peroxidase-labeled monoclonal antibody

F47-9 F67-5 F126-5

Amt (p.g) of unlabeled antibody giving 50% inhibitiona F47-9 F67-5 F126-5

500 >500

>500 125 >500

150 6 40

a Binding of peroxidase-labeled antibodies F47-9 (1/2,500), F67-5 (1/1,500), and F126-5 (1/2,000) to 1 Rg of lysate of POP2136 cells carrying recombinant plasmid pTHL1007, giving an A450 of 1.0.

fusion proteins were expressed amounting to up to 20% of the total cellular protein as estimated from SDS-PAGE (results not shown). The reactivity of monoclonal antibodies F47-9, F67-1, F67-5, and F126-5 to the fusion proteins produced by the various deletion mutants showed that the binding sites for all four antibodies were positioned within the region from amino acids 19 to 95 (19-95 region) of the PRA protein. As a typical example of the reactivity of these four monoclonal antibodies with the various fusion proteins, a Western blot obtained by using F47-9 as a probe is shown in Fig. 3, lanes 4 to 9. Recombinant phage Y3180. Previously, recombinant phage Y3180, expressing an antigen recognized by monoclonal antibody F47-9, was selected from the lambda gtll library of M. leprae by Young et al. (40). This recombinant contained a 4-kbp EcoRI insert. We further analyzed Y3180 and found that the recombinant phage produced a 116K fusion protein that reacted with F47-9 (17; Fig. 3, lane 10). None of the other three monoclonal antibodies reactive to PRA reacted with this 116K fusion protein (results not shown). DNA sequence analysis of the 177-bp EcoRI-BgIlI M. leprae DNA fragment adjacent to the lacZ gene of lambda gtll revealed that the fusion protein expressed by Y3180 consists of 18 amino acid residues in addition to 3-galactosidase (Fig. SA). The DNA sequence of the EcoRI-BglI fragment of Y3180 showed no homology with the sequence we determined with the recombinants pTHL1019 and pTHL1007. Therefore, we conclude that Y3180 and pTHL1019 express polypeptides that are unrelated but contain similar antigenic sites recognized by monoclonal antibody F47-9. DTH reaction to PRA. To determine whether PRA would elicit a delayed-type hypersensitivity (DTH) reaction, we tested the reactivity of purified Cro-p-galactosidase, the Cro-p-galactosidase-PRA recombinant fusion protein, and M. leprae sonic extract in M. leprae-sensitized guinea pigs. Cro-,B-galactosidase gave a strong and about equal reactivity in M. leprae-immunized and in nonimmunized animals (Table 3). Apparently, there is a substantial reactivity to this control antigen independent of immunization with M. leprae. Nevertheless, the PRA fusion protein induced a DTH reac-

M. LEPRAE PROLINE-RICH ANTIGEN

VOL. 58, 1990

TABLE 3. Skin test reactivity of PRA fusion protein in guinea pigs Guinea pigs (n = 5)

M. Ieprae vaccinated Controls

Avg diam (mean ± SD) in skin test after challenge with 10 pg ofa: M.

leprae

Recombinant

Coo

Crto-sia

sonic extract

PRA fusion protein

Galactosidase

6.3 ± 5.9 0.0 ± 0.0

9.9 ± 0.5b 7.9 ± 1.7

7.5 ± 2.1 6.1 ± 1.0

a The diameters of the skin reactions in millimeters were measured 24 h after challenge. Vaccination was done with 2 x 109 whole M. leprae cells 28 days prior to challenge. b Significant (P < 0.05; Student's t test) increase compared with control animals and with control antigen (Cro-3-galactosidase) in same group of animals.

tion in M. leprae-immunized animals which was significantly stronger than the background reactions in both immunized and nonimmunized control groups. This increased response to the recombinant fusion protein suggests that PRA can elicit DTH reactivity. DISCUSSION To characterize the 36K antigen of M. leprae, designated in this study as PRA, we analyzed recombinant DNA clones obtained from two different gene libraries of M. leprae, a lambda gtll library (40) and a cosmid library (8). Initially, we used a panel of four monoclonal antibodies directed to PRA, F47-9, F67-1, F67-5, and F126-5, to select recombinant DNA clones that express PRA antigenic determinants from the lambda gtll library. Four recombinants, represented by THL2004, were selected, and all four seemed identical both at the DNA and protein levels. The presence of identical recombinants is probably due to multiplication of a single recombinant during the amplification step used in the preparation of this library (40). Recombinant THL2004 expressed PRA as a fusion protein with ,-galactosidase with an apparent molecular weight of 143,000, suggesting that 85% of this antigen was encoded. However, DNA sequence analysis revealed that THL2004 contained the complete PRA-coding region and that the fusion protein expressed by this recombinant consists of the complete PRA protein linked to ,-galactosidase by three amino acid residues. By DNA hybridization with the M. leprae DNA insert of THL2004 as a probe, cosmid recombinants were selected that contained the complete pra gene on a 3.0-kbp EcoRI M. leprae DNA fragment. Recombinant pTHL1019 expressing the native PRA protein was obtained by subcloning this 3.0-kbp DNA fragment into an expression vector carrying the lambda PL promoter. This pra gene product had the same electrophoretic mobility as the M. leprae-derived PRA antigen and reacted with all four monoclonal antibodies to this M. leprae antigen. Consequently, we conclude that pTHL1019 carries the complete pra gene encoding the M. leprae 36K antigen. Expression of pra was weak and depended on the presence of the PL promoter. A reason for this weak expression might be that pra is separated from the PL promoter by a stretch of DNA of approximately 1.3 kbp (Fig. 1A). The dependence of expression of pra on the coliphage PL promoter could indicate that the M. leprae-derived transcription signals are not present on pTHL1019, which may be the case if pra is part of an operon. On the other hand, these M. leprae signals may be present on pTHL1019, but they may not be functional in E. coli (8).

85

DNA sequence analysis showed that the structural pra genes from two different M. leprae isolates, present in pTHL1007 and pTHL1019, are identical. This finding is consistent with the assumed conservation of the M. leprae genome inferred from Southern blotting analysis (7). pra encodes a protein of 249 amino acids with a calculated molecular mass of 26,299 daltons, which is considerably lower than the apparent molecular weight of 36,000 estimated by SDS-PAGE. This discrepancy might be caused by the relatively high percentage (17.7%) of prolines in PRA. Proline-rich proteins have been shown to migrate with relatively low mobilities during SDS-PAGE, resulting in overestimation of the molecular weight (5). The antigenic sites recognized by the four monoclonal antibodies used in this study were all mapped in the 19-95 region of PRA. This is consistent with the observed crosscompetition investigated for three of these antibodies. Although these results indicate that these four antibodies recognize closely linked epitopes, they do not recognize an identical epitope, as they differ considerably in reactivity to other mycobacterial species (18; Kolk et al., in preparation). Of these four monoclonal antibodies, only F47-9 reacts specifically with M. leprae. Previously, a monoclonal antibody, ML04, was described which recognized an M. leprae protein antigen of approximately the same apparent molecular weight as that of PRA. This antigen was referred to as a 35K antigen (11). The 35K antigen is probably not identical to PRA, as antibody ML04 did not react with any of the recombinant PRA proteins described in this study (16, 18). Lambda gtll recombinant Y3180, isolated by Young et al. (40), expresses a 116K fusion protein which, like PRA, reacts with monoclonal antibody F47-9 (17). It is therefore possible that although PRA and the fusion protein expressed by Y3180 are not homologous at the DNA level, they show homology at the protein level. Consistently, comparison of the amino acid sequences of the epitope-containing 19-95 region of PRA and the 116K fusion protein revealed a segment of 10 amino acid residues with high homology. The homologous regions were localized at amino acids 31 to 41 of PRA and amino acids -4 to 7 of the fusion protein (Fig. SB). The latter region is composed of four P-galactosidase residues and six M. leprae polypeptide-derived residues. Assuming that this region represents the epitope for F47-9, this suggests that the fusion protein produced by Y3180 contains an artificial epitope created by the fusion of P-galactosidase with a polypeptide unrelated to PRA. This explanation is supported by the reactivity of F47-9 with various synthetic peptides based on the amino acid sequence of the fusion protein of Y3180. Although F47-9 reacted with the M. leprae-derived 18-amino-acid synthetic peptide (15), a much stronger reactivity was observed with a peptide containing in addition the -4 to -1 P-galactosidase amino acid residues

(unpublished observation).

The deduced sequence of PRA reveals a peculiar distribution of amino acid residues in the N-terminal part of the protein. The 1-85 sequence has a relatively high content of the residues proline (43.5%), glycine (14%), and serine (13%). In addition, this N-terminal region contains a number of repeats that are identical or similar to the decapeptide PGGSYPPPPP (Fig. 6A). The C-terminal 85-249 sequence contains a repeat of 23 amino acid residues with 43% homology (Fig. 6B). The biological function of these repeats is unknown. However, it is clear from this study that the N-terminal part of PRA, which contains the proline-rich repeats, is highly immunoreactive. All epitopes of the available four anti-PRA monoclonal antibodies are positioned in

86

INFECT. IMMUN.

THOLE ET AL.

reagents in serological assays instead of the complete M.

A.

.

PPP 7 8 GSNPTPAPPP 18 PGS SGG-YR8SFA 30 PSE LGSAYPPPTA 43 PP VGGSYPPPPP 55 PGGSYPPPPP 65 PGGSYPPPPP 75

.STGAYAPPPP 85

B.

30% 40%

W

30% 50%

90% 100% 100% 80%

101 VTRVLAYVIDNIPATVLWGIGML 123 II1

11111

VTDITQYNVNQYCATQPTGIGML

156 FIG. 6. Repeated sequences of PRA. (A) Repeated sequences in the N-terminal 1-85 region. The numbers at the right indicate the positions of the amino acids in PRA. The percentages of homology of the sequences in the box with the sequence PGGSYPPPPP are indicated. (B) Repeated sequence in the C-terminal 85-249 region. 134

this region. Furthermore, the epitope for one of these monoclonal antibodies, F47-9, is recognized by antibodies in serum samples from most lepromatous leprosy patients (10, 15, 16). High antibody reactivity to regions containing repetitive sequences has also been described for antigens from other pathogens, such as the circumsporozoite antigen of Plasmodium falciparum (23). The presence of repetitive amino acid sequences on mycobacterial proteins is not unique to PRA. Recently, Shinnick (30) described an M. tuberculosis gene specifying a protein of 517 amino acid residues also containing a number of repetitive sequences. The role of this protein in the immune response to mycobacteria is unknown. The overall PRA sequence shows no significant homology with any of the protein sequences from the PIR data bank 'NBRF release 17) as determined with the FASTA program (26). However, the proline-rich 1-85 region of PRA shows significant homology (28 to 48%) with a number of eucaryotic proline-rich proteins such as the collagen alpha chain 1 of human, mouse, rat, bovine, and chicken origin, human proline-rich phosphoproteins, proline-rich proteins (PRPs) of human, mouse, and rat origin, and the Plasmodium circumsporozoite antigen. PRPs, which constitute approximately 70% of the protein content of human saliva, share a number of properties with the 1-85 region of PRA (1). PRPs are characterized by a relatively high content of the amino acid residues proline (25 to 42%), glycine (16 to 22%), and glutamic acid/glutamine (15 to 28%), which make up approximately 70 to 88% of their total amino acid content. The content of two of these residues (proline and glycine) in the 1-85 sequence of PRA is also relatively high (57.5%). However, instead of a high content of glutamic acid/glutamine, PRA contains a relatively high percentage of serine in this region. Furthermore, PRPs and the 1-85 sequence of PRA contain a series of proline-rich repeats. The biological function of PRPs is not very well established, but calcium-binding properties have been demonstrated for some of them. We have not yet studied the ability of PRA to bind calcium. A highly sensitive and specific serological test for lepromatous leprosy has been developed using a competition assay with monoclonal antibody F47-9. The sequence derived in this study and the partial mapping of the epitope recognized by F47-9 will enable us to use synthetic peptides corresponding to a well-defined specific region of PRA as

leprae antigen. The PRA protein when fused to Cro-3-galactosidase evoked a small but significant DTH reactivity in guinea pigs immunized with M. leprae. However, the Cro-p-galactosidase portion of this protein also induced a marked response. This background reactivity could have masked to some extent DTH reactivity to PRA. Experiments using the native recombinant antigen and the M. leprae purified protein are now in progress to determine more accurately the DTH reactivity elicited by PRA. The PRA antigen has already been implicated in the cellular immune response to M. leprae by Ottenhoff and co-workers, who revealed the presence of both helper and suppression-inducing T-cell epitopes on this antigen (24, 25, 36). In future studies synthetic peptides will be used to further characterize the epitopes on PRA that are recognized by antibodies and T cells from individuals infected with M. leprae. Such studies will further elucidate the role of this antigen in the immune response to M. leprae. ACKNOWLEDGMENTS We thank Richard Young for the M. Ieprae genomic library in lambda gtll and recombinant Y3180, Josephine Clark-Curtiss for the M. Ieprae genomic library in pHC79, Hennie Hodemaekers for synthetic oligonucleotides, Ruurd van der Zee for computer analysis of protein sequences, Jan van Embden for critical reading of the manuscript, and Marrie Tensen for designing the figures. This investigation received financial support from the Netherlands Leprosy Relief Association, the Q. M. Gastmann-Wichers Foundation, and the Commission of the European Communities Directorate General for Science, Research and Development (grant TS2-0111-

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